Endothelial cells that line blood vessels pose a barrier to the flow of fluids and blood components to tissues. Different vascular beds have endothelial barriers of varying permeability. Generally microvascular endothelial cells form tighter barriers than macrovascular endothelial cells, which line large vessels that have multiple layers of cells. Vascular leak and edema usually arise when the microvascular endothelial barrier is compromised. While edema can be tolerated in certain peripheral tissues it can be fatal in the brain or the lung where it can increase intracranial pressure in the former and interfere with gas exchange in the latter.
Microvascular endothelial cells open and close the permeability barrier in response to permeability inducing stimuli. One stimulus which has been shown to cause permeability in both the brain microvasculature (BBB) and the pulmonary microvasculature is hypoxia, which can result in acute mountain sickness (AMS).
AMS is characterized by fatigue and headache which is poorly understood but shown to be related to the low oxygen concentration (hypoxia) at altitude. Mild AMS lies at one end of a spectrum that proceeds to the other end delineated by the often fatal high altitude cerebral edema (HACE). Another related and severe consequence of rapid ascent to altitude is high altitude pulmonary edema (HAPE). Even milder symptoms of AMS have been associated with altered vascular permeability which can also be observed in the face, hands, or feet (Schoene, Chest, 2008, 134, 402-416). High altitude-related vascular leak which seems to occur in different systems is not explainable by blood pressure changes (Lewis et al., Eur. J. Clin. Invest., 1997, 27, 64-68).
Several studies have reported increased permeability in the brain, lung or cultured endothelial monolayers in response to hypoxia (Ali et al., Am. J. Physiol., 1999, 277, L1057-1065; Allen et al., Stroke, 2010, 41, 2056-2063; Baudry et al., Am. J. Respir. Crit. Care Med., 1998, 158, 477-483; Carpenter et al., J. Appl. Physiol., 1998, 84, 1048-1054; Carpenter et al., Am. J. Physiol. Lung Cell. Mol. Physiol., 2001, 281, L941-948; Hansen et al., Scand. J. Clin. Lab. Invest., 1996, 56, 367-372; Hassoun et al., Am. J. Respir. Crit. Care Med., 1998, 158, 299-305; Lewis et al., Eur. J. Clin. Invest., 1997, 27, 64-68; Morocz et al., Exp. Neurol., 2001, 168, 96-104; Ogawa et al., J. Clin. Invest., 1990, 85, 1090-1098; Ogawa et al., Am. J. Physiol., 1992, 262, C546-554; Ogawa et al., Adv. Exp. Med. Biol., 1990, 281, 303-312; Partridge, Am. J. Physiol., 1995, 269, L52-58; Pinsky et al., Semin Cell. Biol., 1995, 6, 283-294; Stelzner et al., J. Clin. Invest., 1988, 82, 1840-1847; and Wojciak-Stothard et al., Am. J. Physiol. Lung Cell. Mol. Physiol., 2006, 290, L1173-1182). Fluid and macromolecules cross endothelial barriers either through the cells (transcellular route), or through gaps between these cells (paracellular route). The latter route is believed to be the major contributor to tissue edema (Dudek et al., J. Appl. Physiol., 2001, 91, 1487-1500). Gap formation between cells which determines paracellular permeability is generally believed to be regulated by contractile forces from within the cell counterbalanced by adhesive forces between cells and between cells and the extracellular matrix (Dudek et al., J. Appl. Physiol., 2001, 91, 1487-1500). The changes in biomechanical forces are dynamic. In healthy normally functioning endothelial layers and in response to physiological stimuli, gaps form transiently and their decrease correlates with barrier augmentation and decreased permeability.
In endothelial cells, hypoxia causes activation of the ROCK-MLCP-MLC2 pathway leading to increased permeability. Hypoxia also causes activation of the p38-MK2-HSP27 pathway which correlates with decreased permeability. The increased contractility of cells is generally believed to be due to interaction of actin filaments with myosin. This process has been shown to be regulated by myosin light chain (MLC2) phosphorylation (Garcia et al., J. Cell. Physiol., 1995, 163, 510-522), which in turn is regulated by kinases and phosphatases, such as myosin light chain phosphatase (MLCP). The increased phosphorylation of MLC2 and ensuing contraction of endothelial cells promote formation of gaps between endothelial cells and increase permeability. Stimuli that weaken the endothelial barrier, e.g., sodium fluoride, act through Rho-activated kinase (ROCK) (Wang et al., Am. J. Physiol. Lung Cell. Mol. Physiol., 2001, 281, L1472-1483). ROCK increases MLC2 phosphorylation through phosphorylating MYPT1, a subunit of MLCP leading to the latter's inhibition (Essler et al., J. Biol. Chem., 1998, 273, 21867-21874; and Robertson et al., Br. J. Pharmacol., 2000, 131, 5-9). MLC2 phosphorylation has been correlated with increased endothelial permeability in response to a variety of agents and stresses including hypoxia. ROCK has been proposed to mediate hypoxia-induced smooth muscle contraction via phosphorylating MYPT1 (Wang et al., Am. J. Respir. Cell. Mol. Biol., 2001, 25, 628-635; and Wang et al., Am. J. Respir. Cell. Mol. Biol., 2003, 24, 24). Another report on porcine pulmonary artery endothelial cells suggested that hypoxia induced Rho activation mediates increased barrier permeability (Wojciak-Stothard et al., Am. J. Physiol. Lung Cell. Mol. Physiol., 2005, 288, L749-760). Recently, hypoxia-induced brain endothelial barrier dysfunction has been linked to activation of Rho and ROCK (Allen et al., Stroke, 2010, 41, 2056-2063) Inhibition of ROCK blocks hypoxia-induced permeability of endothelial monolayers (Liu et al., J. Cell. Physiol., 2009, 220, 600-610). Hypoxia and TGFβ increase the phosphorylation of MYPT1 and MLC2 in rat pulmonary endothelial cells (RPMEC) (Liu et al., J. Cell. Physiol., 2009, 220, 600-610). These results describe that hypoxia increases endothelial barrier permeability through activating ROCK-MLCP-MLC2 signaling and endothelial contractility (Liu et al., J. Cell. Physiol., 2009, 220, 600-610). Furthermore, hypoxia causes a transient increase in endothelial cell contractility that can be blocked by inhibiting ROCK, but not by inhibiting p38 (An et al., Am. J. Physiol. Cell. Physiol., 2005, 289, C521-530).
One of the most commonly reported effects related to increased permeability is increased actin stress fiber formation, typically associated with contractility. Several agents that induce endothelial permeability, e.g., thrombin and H2O2, have been associated with increased actin stress fiber formation. Hypoxia can induce actin stress fiber formation in RPMEC via activation of p38 and MK2 and phosphorylation of HSP27 (Kayyali et al., J. Biol. Chem., 2002, 277, 42596-42602). However, recent research suggests that instead of being associated with increased permeability, this actin stress fiber formation is actually associated with decreased permeability and a stronger barrier (Liu et al., J. Cell. Physiol., 2009, 220, 600-610).
p38 MAP kinase is implicated in stress response, and when activated by hypoxia, it phosphorylates and activates the kinase MK2, which then phosphorylates the small heat shock protein HSP27 leading to actin filament formation in endothelial cells (An et al., Am. J. Physiol. Cell. Physiol., 2005, 289, C521-530; and Kayyali et al., J. Biol. Chem., 2002, 277, 42596-42602). Unphosphorylated HSP27 binds actin and blocks its polymerization, and has been associated with decreased actin stress fibers and decreased focal adhesions (Schneider et al., J. Cell. Physiol., 1998, 177, 575-584).
Consistent with the role of HSP27 phosphorylation in mediating hypoxia-induced stress fibers, RPMECs that are stably transfected with phospho-mimicking (pm) HSP27 contain significantly more actin stress fibers than mock-transfected RPMEC, a phenotype that could be rescued by siRNA against the pmHSP27 (Kayyali et al., J. Biol. Chem., 2002, 277, 42596-42602; and Liu et al., J. Cell. Physiol., 2009, 220, 600-610). Furthermore, the RPMEC transfected with pmHSP27 form a tighter permeability barrier than wild type RPMEC (Liu et al., J. Cell. Physiol., 2009, 220, 600-610).
Inhibiting the actin stress fibers in pmHSP27 overexpressing cells with cytochalasin D abolished the reduced monolayer intercellular gaps and permeability (Liu et al., J. Cell. Physiol., 2009, 220, 600-610). Increased contractility is dissociated from p38-MK2-HSP27-mediated stress fiber formation which instead is associated with increased cell adhesiveness (An et al., Am. J. Physiol. Cell. Physiol., 2005, 289, C521-530).
Increased understanding of signaling pathways in normal physiology and disease led to numerous new drug candidates that target these signaling pathways. The majority of these drug candidates are designed to be inhibitors of enzymes, such as kinases, because they are easier to develop based on better knowledge of the structure of active sites or substrate binding sites. For example, inhibitors of p38 have been developed by several drug companies to target inflammation and other processes. These inhibitors have not moved to the clinic as fast as anticipated because of their toxicity. Indeed, one major toxicity associated with p38 inhibitors is related to intestinal epithelial permeability barrier compromise (Morris et al., Toxicol. Pathol., 2010, 38, 606-618). The fact that MK2 inhibitors caused a similar type of toxicity indicates that the effects on epithelial barrier function might be due to a role for p38-MK2 signaling in proper barrier function (Morris et al., Toxicol. Pathol., 2010, 38, 606-618).
MK2 is the immediate kinase that phosphorylates HSP27. Elucidation of the structure of MK2 revealed that it contains an autoinhibitory domain. This domain was reported to be a target of Kaposin (Kaposin proposed to bind to and block the region of MK2 where the autoinhibitory domain binds), the product of the HSV virus that causes Kaposi's sarcoma (McCormick et al., Science, 2005, 307, 739-741; and McCormick et al., J. Virol., 2006, 80, 6165-6170).
Transduction domains have been reported to increase the penetration of peptides into cells. One sequence with demonstrated effectiveness has been identified (Tessier et al., J. Vasc. Surg., 2004, 40, 106-114).
Pulmonary edema is a condition caused by excess fluid in the lungs. This fluid collects in the numerous air sacs in the lungs, making it difficult to breathe. In most cases, heart problems cause pulmonary edema. Fluid can accumulate for other reasons, however, including pneumonia, exposure to certain toxins or infections, adverse reaction to medications, severe injuries (trauma), systemic infection (sepsis), pneumonia and shock, and exercising or living at high elevations. Depending on the cause, pulmonary edema symptoms may appear suddenly or develop slowly.
Treatments include morphine (Astramorph, Roxanol), afterload reducers (e.g., nitroprusside (Nitropress), enalapril (Vasotec) and captopril (Capoten), and blood pressure medications. Pulmonary edema can be fatal, even if treated.
Additional treatments for augmenting permeability barriers and treating diseases, conditions, disorders, and/or injuries associated therewith, such as pulmonary edema and other lung diseases and injuries are needed.